Method using immobilized yeast to produce ethanol and alcoholic beverages

ABSTRACT

According to the invention, yeast cells are immobilized on a substantially noncompressible carrier having anion exchange properties. The immobilized yeast can then be used to ferment a sugar-containing substrate. The noncompressibility enables the system to be sterilized and to operate under pressure. A preferred example of the carrier is granular DEAE cellulose.

This invention relates to production of ethanol and more specifically toproduction of alcoholic beverages using immobilized yeast which is boundto a regenerable carrier material. Yeast immobilization can be carriedout in the fermentation reactor itself thus minimizing the risk ofcontamination during the immobilization.

BACKGROUND OF THE INVENTION

Fermentation is an art that began before recorded history. It was,however, only in 1866 that Pasteur published his works on winefermentation, analyzed the reasons for spoilage, and prescribed anappropriate treatment for wine making.

Traditionally the fermentation takes place with the wild yeasts thatoccur on all fruits and berries. Fermentation with pure culture yeastsis used more often today in order to consistently produce good qualitywines. All these general aspects of wine making and characterizations ofthe yeasts are presented in "Biotechnology" edited by H-J Rehm and GReed Vol. Food and Feed Production with Micro-organisms, Verlag Chemieor in Prescott & Dunn's Industrial Microbiology 4th edition, chapter 9,1982 edited by G. Reed, the disclosure of which in incorporated hereinby reference.

Traditional batch fermentation is time consuming. A typical time for thealcohol fermentation in wine making normally requires at least 20-50days. Although a continuous process with suspended yeast might bethought to speed this process it is difficult to operate and maintainfree from microbial contamination. Moreover, to speed up thefermentation rate, the yeast cell concentration in the fermenting mustshould be increased while on the other hand the ethanol inhibitionshould be minimized. In a batch fermentation, for example with applejuice, the yeast cell concentration is 1-2 ×10⁸ cells/ml of must/broth.

A higher yeast cell concentration can, for example, be arranged byimmobilizing the yeast on a suitable carrier packed in a column. Whenthe fermenting must is passed through the column packed with immobilizedyeast, the number of yeast cells in contact with the volume of must inthe reactor is greatly increased. This greater contact area results in afaster fermentation. The column reactor also reduces the ethanolinhibition since the ethanol containing product is continuously removedfrom the yeast bed. This reduction is greater as an ideal plug flowsituation is approached.

It is known that yeast cells can be entrapped in calcium alginate andthe resulting immobilized yeast can be used for fast fermentations.Numerous literature references describe this technique as it is appliedusually in laboratory scale. Some efforts, however, have also been madeto commercialize this technique. One of the best known is probably thatof Kyowa Hakko in Japan, where alginate immobilized yeast is used forfeed ethanol production.

For commercial scale operations, a major difficulty with alginateentrapment is the manner in which the particles are formed. It must becarried out at the production site where a yeast slurry and a solutionof sodium algonate are mixed together. When this mixture is then fedinto a calcium-salt solution, the alginate precipitates and at the sametime occludes the yeast cells within the precipitated particles. Theparticles usually are in the form of droplets/beads.

A process plant that utilizes alginate entrapped yeast must havespecially designed equipment just to produce these beads. Furthermore,there is a potential risk for contaminating the yeast with wildmicroorganisms. This is especially critical when production of analcoholic beverage flavor is important (e.g., beer, wine, cider andsimilar kinds of products). For technical/fuel ethanol production thecriteria of contamination is not as important as in the consumablesalthough it does affect the productivity.

A second major difficulty for alginate beads used on a commercial scaleis in the physical strength of the beads. The beads are soft and easilycompressible. Operating large fermentation columns can be a problem andfast downflow process streams are difficult to handle. On the otherhand, a typical upflow mode in fermentation greatly wears the beads.Also, to run a reactor downflow under pressure with compressiblematerial is virtually impossible.

A third difficulty with entrapment is the diffusion limitations whichslow down the accessibility of the substrate in contact with the yeastinside the bead.

Finally, if the system becomes contaminated or otherwise disturbed sothat a continuous operation must be discontinued, the whole lot ofcolumn material (alginate together with the yeast) must be discarded. Noreuse is possible.

Therefore, it is an object of the present invention to develop a methodfor continuous column fermentation that does not require on-siteformation of the column material. Another object is to develop a methodthat eliminates sources of contamination. Yet another object is todevelop a method of this type that can withstand pressure. Still anotherobject of this invention is to develop a method where the carriermaterial can be regenerated and reused. Further objects includedevelopment of processes that produce alcohol at reasonably rapid ratessuch as at least a minimum of about 1 bed volume of about 5 percentalcohol per day.

SUMMARY OF THE INVENTION

These and other objects are achieved by the present invention which isdirected to a method for the primary production of an ethanolic product.According to this method, an aqueous substrate containing a dissolvedyeast-fermentable carbohydrate is contacted with yeast immobilized upona carrier having anionic exchange properties. The contact can bepreferably accomplished by passing the aqueous substrate through apacked bed reactor of the yeast immobilized upon the carrier. However,it may also be accomplished by use of the immobilized yeast in afluidized bed reactor.

The yeast-carrier combination is comprised of yeast bound to thesurfaces of the porous carrier. The carrier is substantiallynon-compressible. It is composed of a continuous porous matrix, oralternatively, of dimpled or reticulated, porous particles. The matrixor particles, in turn, are composed of individual microparticles ormicrofibers. This carrier structure provides a maximal surface area forcarrying yeast cells. The resulting high number of yeast cells per unitvolume of carrier makes rapid fermentation possible.

The particulate or matrix character of the carrier is produced byloosely binding, felting, weaving, gluing or agglomerating (hereinafterbinding) the microparticles or microfibers together. The binding isaccomplished by establishing chemical, adherent or mechanical links atsome of the contact points between the individual microparticles ormicrofibers. Chemical binding is accomplished by causing a chemicalcross-linking reaction at these points. Adherent ending is accomplishedby agglomerating or gluing the microfibers or microparticles togetherthrough the use of an additional ingredient such as a thermoplasticresin. Mechanical binding is accomplished by entangling or knotting thefibers at the contact points or by joining the particles by meshingtheir surfaces together. In its final form, the matrix will comprise acontinuous structure throughout the reactor, much like cotton fluff orfilter paper packed into a tube. Also, in their final form, theparticles will be discrete and individual.

The microfibers or microparticles are composed of any anion exchangesubstance that can be formed into the desired, rough-surfacedmicrofibers or microparticles. These substances include native orregenerated cellulose or rayon that is derivatized to provide anionexchange character; synthetic anion exchange resins such asphenolformaldehyde resins, and agarose or dextrin based anion exchangeresins. The preferred carrier is a porous, particulate anion exchangeresin derived from cellulose or rayon that has been chemically modifiedto provide anion exchange character. Especially preferred embodimentsinclude microfibers or microparticles of diethylaminoethylenesubstituted cellulose, adherently bound by agglomeration withpolysterene.

It is believed that the electric forces established between thepositively charged resin and the negatively charged yeast cells areprimarily responsible for the binding of yeast cells to the surfaces ofthe resin. This binding minimizes substantial leaching of the yeast yetpermits intimate contact between the yeast and the aqueous medium.

The aqueous substrate used as a starting material is composed at leastof water and a fermentable carbohydrate such as hydrolyzed starch,sucrose, glucose, fructose, maltose or maltotriose (lactose and xylose).The concentration of sugar will be sufficient to permit continuousproduction of alcohol but will be not so high that the fermentiveactivity of the yeast is completely inhibited.

According to the method of the invention, it is important to control theprocessing parameter involving carbon dioxide produced by thefermentation. The carbon dioxide can be maintained in the fluid productstream or removed. In a preferred method, a series of columns isemployed, the interconnections of which are adapted for removal of gasfrom the column outlet stream. By maintaining the columns under somepressure, a carbonated product can be produced. If the columninterconnection taps are all employed, a product without carbonationwill be produced. The column pressure, however, cannot be so high as tosubstantially diminish the yeast fermentation. Facile routineering canestablish this limit which, in general, will be at least about 14 bar.

In addition to pressure, other processing parameters can be varied toaffect the output of ethanol and taste of the product. These parametersinclude column temperature, aqueous substrate feed rate, columnresidence time, direction of substrate flow in the column (with oragainst gravity), periodic reversal of flow direction, yeast strain,yeast concentration and yeast nutrients in the aqueous substrate.Suitable ranges for these parameters include a temperature of about 0°to 40° C., feed rate 0.01-10 reactor bed volume (BV)/hour, a residencetime of about 0.1-100 h and yeast concentration of about 109 to about1012 yeast cells per litre carrier Yeasts such as Saccharomyces orCandida can be used. Generally, these parameters will be adjusted toproduce an ethanol concentration of from 0.05 to 15 percent.

In a preferred method of the invention, a slurry of the particulatecarrier is first placed in a column and the carrier allowed to settleinto a packed column. The carrier is sterilized by a method such aswashing with hot caustic. After neutralizing with sterile, dilute acidicsolution and rinsing with sterile water, the column is then eluted witha yeast broth so that the yeast becomes attached to and immobilized onthe carrier particles. After this pretreatment, the column of yeast isused as described above.

In a further preferred method of the invention, an ethanolic productthat is consumable is produced. This product can be wine, sake, analcoholic fruit, berry or vegetable drink, carbonated versions thereof,beer, or low alcohol version's of the foregoing products.

The aqueous substrate used to produce the consumable product is derivedfrom a source such as a fruit, berry or vegetable juice or extract, awort, hydrolyzed plant material or an aqueous syrup containing afermentable sugar derived from a natural or synthetic source. The juicewill be a liquid pressed from a fruit, berry or vegetable. The extractwill be a liquid produced by combining the fruit, berry or vegetablewith water and processing by mashing, cooking, pressing, mixing and thelike. The hydrolyzed plant material will be material that is derivedfrom cellulose, hemicellulose and/or starch through a technique such asacid, enzyme or auto-hydrolysis.

Another method of the invention is directed to production of low alcoholbeverages such as those containing less than 0.2 percent by volumealcohol. According to this method, the steps practiced and theyeast-carrier material are the same as those of the primary productionmethod. The sugar content and feed rate of aqueous substrate, however,are modified to provide the "low alcohol" production. The feed rate isincreased to an extent that decreases the alcohol concentration to thevalue desired. The sugar concentration is also appropriately adjusted sothat the beverage is not overly sweet yet enough sugar is present forfermentation to alcohol.

The invention is further directed to the combination of the carrier andyeast in any form. Preferred forms include that made in situ, asdescribed above, and a dried combination.

The dried combination in aseptic condition can be packaged, transportedto a fermentation plant and reconstituted for by immersion in aqueousnutrient. Alternatively, the dried form can be packaged for home use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a simplfied flow scheme for preparing the immobilized yeastcolumns.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is based upon a yeast-carrier system, describedabove, that is non-compressible and sterilizable An especially preferredexample of a carrier for use in producing the yeast-carrier system isparticulate DEAE cellulose (weak anion exchanger) agglomerated withpolystyrene. This carrier is commercially available and is well-knownfor immobilizing enzymes, typically isomerase. (See U.S. Pat. No.4,355,117 for details regarding this carrier.)

In a preferred process for preparing the yeast-carrier system asdepicted in FIG. 1, the dry carrier is hydrated in water and pumped as aslurry into a column reactor. The preferred DEAE cellulose carrier isstable in acid and alkaline media and can withstand a temperature up to100° C. It can thus be easily sterilized in the column, for example,with hot caustic. Other resins can appropriately be sterilized insimilar fashion.

Yeast immobilization onto the carrier is accomplished aftersterilization of the carrier. It is carried out by pumping a cultured,active yeast slurry into the reactor. The carrier then adsorbs theyeast. The yeast cells attach to the carrier surfaces as a result of theoptimal surface shapes of the carrier. It is rough, reticulated andexists as a continuous or particulate porous matrix. The attachment isbelieved to result from electrical forces between the anionic exchangegroups (positively charged) of the carrier and the negatively chargedyeast cell wall.

Preferably, the yeast cells are grown on the carrier to provide adensity of between about 10⁹ -10¹² cells per liter of carrier, with adensity of about 10⁹ cells being particularly preferred. The densitymust be sufficient to provide enough enzymatic activity to substantiallyconvert the sugars in the aqueous substrate to ethanol.

The carrier is important in terms of providing an adequate environmentfor yeast growth and contact with the aqueous substrate. Thenon-compressible anion exchange carrier, such as agglomeratedDEAE-cellulose, has several beneficial qualities compared with soft,gel-like carriers, such as alginate beads. These qualities include fewermass transfer problems, easier immobilization, faster start-up, easierscale-up, much improved regenerability and a long life-time of thecarrier.

In laboratory scale experiments a gel-like carrier, i.e., alginatebeads, and a preferred carrier of the invention, i.e., DEAE-cellulosegranules, have been compared. The carrier made from DEAE-cellulose wasmore facile to use for ethanol production and with many substrates had amore desirable production capacity These factors suggest a bettercontact between yeast and aqueous substrate during treatment.

Although not intended as a limitation of the invention, it is believedthat the mode of interaction of the immobilized yeast and the carrieraccording to the invention may explain the increased yield. Electronmicrographs show that in the alginate beads, the yeast grows incolonies, some

of which grow through the alginate layer to the bead surface. The yeastcolonies presumably act as the "active sites" in the alginate beads. Themicrophotographs show that the DEAE-cellulose granules are porous,reticulated matrices of microfibers. This structure permits the aqueoussubstrate to reach yeast cells growing inside the granules. Thus, theyeast grows rather loosely and separately in the internal and externalpockets of the microfibers and these individual cells act as "activefermentation sites" in the column. Accordingly, more yeast cells perunit carrier surface area are available for fermentation when thecarrier of the invention is used.

The mechanism of the yeast cell immobilization onto the surface of thecarrier, such as granular DEAE cellulose, offers many benefits. First,the yeast cells are substantially all on the surface of the carrier.Thus, the cells act as if they were freely suspended in a solution.

Second, there are no substantial diffusion limitations and thefermentable fluid substrate can freely come into contact with the yeast.Also the nutrients that the yeast needs for life are available withouthindrance from the need to permeate into the interior of the carrierparticles.

Third, the carrier properties according to the invention also permiteasy start-up and regeneration because the yeast cell immobilization cantake place in situ within the column. This procedure lowers the risk ofcontamination and improves the conditions of the system as a whole. Inaddition, the column can be easily regenerated which is an importanteconomic feature. Regeneration is sometimes expedient because ofchemical impurities or contamination from the aqueous, microbialcontamination or from mutations of the immobilization yeast itself. Toregenerate a column reactor made from a carrier such as DEAE cellulose,for example, the spent column can be washed and sterilized with a hotcaustic solution or

with another sterilizing medium such as an organic agent in water andthe like. After washing and neutralization, the carrier is ready forrepitching and reimmobilization. In pilot scale set-ups, such a columnhas been utilized for at least thirteen weeks without need forregeneration. In lab scale, a similar column has been utilized for about30 weeks.

In general, the entire reactor system, including the carrier, can besterilized with the above-mentioned treatment. When an asepticallycultured yeast is then pumped or eluted through the carrier bed, therisk of contaminating the yeast/carrier with wild strains or bacteriawill be minimal. When the yeast is attached to the column (a suitablecharge is about 10⁹ to 10¹² yeast cells/liter carrier) the column can befurther conditioned by slowly pumping a nutrient solution such as anaqueous medium of ammonium phosphate and sugar through the reactor forapproximately one day. This process causes the yeast to flourish to amaximum density.

As is generally true of all yeast-carrier systems

used according to the invention, one of the preferred carrierembodiments, granulated DEAE cellulose, is non-compressible(non-swelling). This property allows much variation as to how thefermentation can be conducted.

Under normal atmospheric pressure, the reactor/continuous fermenter maybe operated as follows. The substrate is fed in from the bottom of thereactor and the CO₂, which is formed during the fermentation, is freelyallowed to evolve from the carrier bed. With this technique, however, anideal plug flow situation cannot be reached (CO₂ bubbles always disturbthe bed) and backmixing causes ethanol inhibition and thus lowerproductivity.

The non-compressible carrier system of the present invention can also beoperated under pressure in order to maintain the carbon dioxide in adissolved state. The column can then also be run in a downflow conditionand in this packed bed system, an ideal plug flow is reached.

Several pressurized columns may also be run in series to avoid highpressure. The series arrangement is especially useful if a largeconcentration of alcohol in the final product is desired. This methodenables the operation in a downflow mode without the harmful release ofcarbon dioxide which could disrupt the packed character of the columni.e. cause channelling. Between the columns, carbon dioxide is separatedfrom the stream. The reactor size can also be kept smaller as no extrafluidization and carbon dioxide-separation space is needed.

Basically any fermentable fluid substrate can be used as feed materialfor production of the ethanolic product provided that any particulateimpurities are filtered out before feeding into the immobilized yeastcolumn. Examples of typical substrates would be:

(i) wort

(ii) fruit juice,

(iii) berry juice

(iv) sugar syrup

(v) starch syrup

(vi) any hydrolysate out of plant material

(vii) sugar syrups flavored by any fruit, berry, malt or similarextracts

(viii) any aqueous substrate that is used in beer, wine or liquorproduction

A typical total sugar concentration would be 100-250 g/L including bothoriginal and added sugars depending on the basic raw material. The yeastnutrients (including sources of phosphorus, nitrogen etc.) need to bebalanced unless they are favorable in the original raw material. Thesugar concentration can, of course, be varied widely depending on howmuch alcohol and how much sweetness the end product is expected to have.It may also contain natural flavors, oils and the like. The sugar willbe present at a concentration of from at least about 1 percent by weightto an amount that will inhibit fermentation function (e.g., about 30 to40 percent by weight), preferably about 4 to 25 percent by weightrelative to the total weight of the medium.

The flow rate of the aqueous substrate through the column is animportant parameter because the ethanol production capacity is dependenton the flow rate. It has also been shown that the flow rate determinesthe number of yeast cells which remain bound in the carrier. With highflow rates, the yeast cell outflow is increased; consequently ethanolproduction may also be dependent on the cell number bound in theimmobilized system, and not only on the throughput rate of thesubstrate. If the flow rate is too high, the ethanol production isincomplete and/or inadequate; by controlling the flow rate, the ethanolconcentration can be controlled, typically by seeking a concentrationwhich is from about 0.05 percent to about 15 percent by volume relativeto the total volume of the aqueous substrate. By adjusting the flowrate, the fermenter can obtain an ethanol concentration within thisrange.

The amount of yeast cells which remain bound to the carrier materialappears to be relatively stable at low flow rates, suggesting that yeastgrowth and leaching of the cells are balanced. With higher flow ratesleaching increases but the cell number returns back to the normal levelagain when the flow rate is decreased. The flow rate should be highenough to enable the leaching of dead cells in order to avoid autolysis.During the operation of the immobilized yeast column, the yeast ismaintained viable by fermentable sugars in the aqueous substrate.

The immobilized yeast column reactor can be pressurized as discussedabove. The operation pressure should be high enough to keep the carbondioxide soluble in the column and to avoid the possible channeling ofthe CO₂ bubbles through the immobilized yeast column. Typical measure is14 bar.

A suitable temperature is that used in traditional batch fermentationprocesses. It will range from about 0 to about 40° C., preferably about10° to about 35° C. This temperature can be maintained either by warmingthe columns with heating jackets or maintaining the columns in anenvironmentally controlled room, or by cooling. Of course, the yeastalso develops a certain degree of heat that also can be used toadvantage. The pressure is chosen according to the operationtemperature.

Following fermentation, the eluted ethanolic product is cooled down tothe storage temperature and collected into a buffer tank for theconventional post-fermentation treatment, e.g., filtering,pasteurization and packaging.

The ethanol production system described above is a faster and moreconvenient system than traditional fermentation processes and produces aproduct with acceptable taste equivalent to the taste resulting fromconventional fermentation processes. The time needed for fermentation isreduced from weeks to a matter of hours by the present invention which,along with the continuous nature of the process, provides a vastpotential for time and money savings in commercial alcohol or alcoholicbeverages production.

The following examples further illustrate many aspects of thisinvention. The examples, however, are not meant to stand as limitationsor characterizations of the invention as it has been fully characterizedin the foregoing text.

Preparation of Column Reactor

Granular DEAE-cellulose (GDC) manufactured according to U.S. Pat. No.4,355,117 by Finnish Sugar Co. Ltd. of particle size 315-840 m or470-840 m was used as a carrier for the examples 1-3. In all theexperiments the carrier was filled, sterilized and yeast immobilizedthereon according to the following procedure for handling GDC:

Procedure for Preparing GDC Column and Immobilizing Yeast

With reference to FIG. 1, the hydration vessel (1) is first filled halfwith water The mixer is started and dry carrier (GDC) is transferred tothe vessel (1). When the hydration is completed (about 5 hours) theimmobilized yeast reactor (2) is filled half with water and the carrierwater slurry from the hydration vessel (1) is transferred to the reactor(2). In order to maintain the water level in the reactor the bottomvalve on the reactor is adjusted so that inlet and outlet flows from thereactor are about the same. The carrier in the reactor is thensterilized with dilute caustic (3) by pumping it through the reactor(2). The carrier bed is then rinsed with water and neutralized bypumping a suitable dilute acid (4) through the carrier bed in thereactor (2) and finally the carrier is rinsed with sterile water.

A yeast slurry is made up in the vessel (5). The yeast slurry is thenpumped through the carrier bed in about 1-4 hours and the yeast willbind itself to the carrier. The yeast is now immobilized onto thecarrier and the reactor (2) is basically ready for fermentation.

The substrate from vessel (6) is then pumped through the reactor eitherthrough the bottom or top inlet. By adjusting the flow rate one cancontrol the level of fermentation. Slow flow rate (about lBV/day) meanslong contact time between the carrier and the substrate and thus highalcohol levels. Faster flow again lowers the alcohol level. The reactorcan be run under atmospheric pressure feeding from the bottom inlet letthe carbon dioxide separated from the system freely through a separationport (7). The product is collected to a receiving vessel (8) from whereit is further processed through filtration etc. before final bottling.

When the system is operated under pressure, the substrate (6) is fedfrom the top inlet in order to keep the carrier bed in packed form andthus reach a plug flow. The fermentation rate is controlled in such amanner (by flow rate) that the applied pressure is high enough tomaintain the formed carbon dioxide dissolved. The pressure is releasedthrough valve (9) on the receiving vessel unless the final product isintended to contain carbon dioxide. In order to keep pressure anddissolved carbon dioxide in reasonable limits, it is advisable to havetwo or more similar reactors (2) in series and release the excess carbondioxide between each reactor.

EXAMPLE 1 Wine

Apple juice has been used as substrate in four different column reactorfermentation experiments.

In the first two experiments (A₁ and A₂) the yeast was normally culturedas it is done for batch fermentations and the carrier beds of about 500ml each were prepared with an amount of yeast cells as described above.

Immediately after the yeast had been rinsed through the carrier bed inthe two columns (diameter - 50 mm and height - 250 - 300 mm) thesubstrate feed was started with a flow rate of 1 BV/day. The substratewas pasteurized apple juice where 1 of ammonium phosphate was added asyeast nutrient. The total sugar concentration was adjusted to 220 g/1 byadding sugar to the apple juice. The fermentation started slowly andafter 3 weeks the outflowing wine had 4.5-6.5 % of ethanol. The carrierin this experiment had a particle size of 0.315 to 0.840 mm.

Carbon dioxide removal from the column was complicated by the narrowshape of the columns. A column, which was loaded with a carrier of theparticle size 0.470-0.840 mm, behaved better as carbon dioxide couldmore easily percolate out from the carrier/yeast bed. From both columnssome GDC was blown out with the carbon dioxide bubbles 50 days operationthe flow rate was reduce to about 0.8 BV/day which immediately increasedthe ethanol level during the following week from 5 % to 7-8 %. After 75days of operation the nutrients were further increased to 2 g/1 but noeffect could be seen in the columns A₁ or A₂.

The third experiment A₃ was carried out in column which was wider andshorter. Reason for this was to make carbon dioxide removal easier. Whenthe carrier bed had been sterilized with hot caustic and washed andneutralized with water and sodium-metabisulphite, the yeastimmobilization was carried out with substantially higher yeast cellconcentration For 500 ml of GDC, 600 ml of yeast slurry was used whichcontained 150 million cells/ml. The leakage through the column was 5million cells/ml so the total mobilized amount was 9×10¹⁰ cells. This isequal to 2×10⁹ cells/ml GDC or 5×10⁹ cells/g GDC.

For one day the column was eluted with yeast nutrient solution beforebeginning with apple juice substrate. The fermentation started rapidlywhen compared to A₁ and A₂ and on the second day the ethanol level wasalready at 8%. The flow rate was 1 BV/day and nutrients 1 g/1 juice.Some material was also lost from this column by carbon dioxide reflex.

Reduction of feed rate after 3 weeks caused the alcohol level to raiseagain. After 1.5 months the nutrient level was raised to 2 g/1 whichraised the alcohol level further and the system stabilized at about a10% ethanol level during its third month of operation.

The fourth experiment was carried out in a 8 1 reactor following thesame procedure as with A₃. The reactor is constructed so that in theoutlet line there is also a screen plate to hold back any GDC particles.The system stabilized into a 10% alcohol level within a week. A portionof the product from the 8 1 reactor was further fed to a second 500 mlcolumn to increase the level of ethanol.

All the experiments were carried out in room temperature 20°-23° C. Whenthe second reactor is operated under pressure high enough to keep thecarbon dioxide dissolved the product is a sparkling champagne type wine,which if bottled under the same pressure as it is fermented makes thesecondary, fermentation unnecessary in the bottles and thus makes the`champagne` process less complicated.

EXAMPLE 2 Beer

In this example, primary beer fermentation can be carried out in animmobilized yeast column.

The column preparation and yeast immobilization can be carried outfollowing the procedure described in the foregoing section on thereactor preparation. A 500 ml carrier bed can be packed in a glasscolumn which is wider in the upper end in order to ease the carbondioxide separation. The wort can be fed into the bottom of thefermentation column. The preferred yeast cell concentration can beadjusted to be 10⁹ cells/g GDC.

Traditional wort for lager beer brewing can be used as a feed materialfor this experiment. The wort can be produced from 18 kg of barley malt(pilsner type) which will give a final volume of 100 1 of finished wort(12.0 P). The mixture of water and malt grist can be mashed in onevessel by programmed infusion method with rests at temperature 48° C.for 15 minutes, 63° C. for 30 minutes, 72° C. for 20 minutes and 78° C.at the end of mashing.

The wort can be clarified in a lautertun and rinsed twice with 78° C.water. The wort can be boiled in a wort kettle for about 90 minutes. Hoppellets can be added at the beginning of boiling (total amount of alphaacids is about 10g). Any precipitation formed during boiling can beseparated in a whirlpool. The clarified wort can be cooled in a plateheat exchanger from 100° C. to 10° C.

The preferred composition of the wort that is sought

    ______________________________________                                        Original extract   12.0       ° Plato                                  Color              10.0       ° EBC                                    Bitterness         25           EBU                                           pH                  5.3                                                       Apparent attenuation rate                                                                        85%                                                        The composition of the wort, however, can vary as follows:                    Original extract    6-18      ° Plato                                  pH                  4.5-5.5                                                   Apparent attenuation rate                                                                        65-100%                                                    ______________________________________                                    

Fermentation of the wort in the reactor column can be started slowly.After one week's operation, the rate will be capable of producing anacceptable beer at less than one bed volume/day feed rate. The columnbehavior was similar to that of the wine reactors A₁ and A₂ in Example1.

Example 3 Sake

In this example low alcohol sake (Japanese rice wine) was produced. Thecolumn was prepared and the yeast was again immobilized following theprocedure set forth in the foregoing section on reactor preparation.

To make the substrate, the rice was first liquefied by steaming andhydrolyzing with alpha-amylase and amyloglucosidase. The resultinghydrolysate was filtered in order to remove all mechanical impuritiesand unhydrolyzed residues. The substrate was finally diluted so that theglucose concentration was 23% when it was fed into the column. Thecolumn had a carrier volume of about 500 ml. Column diameter was 70 mmand diameter to height ratio of carrier about 1:2. Comparison wascarried out with Ca-alginate entrapped yeast. The feed rates, parametersand results for both fermentations are given in the following table.

    ______________________________________                                        Result                                                                        Support     GDC Reactor  Ca-alginate Reactor                                  ______________________________________                                        Fermentation                                                                              15           25                                                   period (day)                                                                  Temperature 15-20        15-20                                                Mean of ethanol                                                                           9.99         13.6                                                 (v/v %)                                                                       Mean of flow rate                                                                         0.069        0.099                                                (BV/h)                                                                        Productivity                                                                              5.44         10.62                                                (g/h/1-support)                                                               ______________________________________                                    

EXAMPLE 4 Low-Alcohol Beverage

The production of a traditional Scandinavian low-alcohol beverage in animmobilized yeast system is described in this example.

By regulating the feed rate in the fermentation reactor column, thefermentation rate can be controlled and the required flavor and alcoholcontent in the product can be achieved.

The column preparation and yeast immobilization can be carried outfollowing the procedure described in the foregoing section on reactorpreparation. The yeast cell concentration is adapted to provide 10₉cells/g GDC.

The aqueous substrate for fermentation can be formulated as follows:

    ______________________________________                                        Crystal (white) sugar   500 g                                                 Honey                   125 g                                                 Soft brown sugar        625 g                                                 2 lemons' juice                                                               Water                    10 l                                                 ______________________________________                                    

A 500 ml carrier bed in a glass column can be eluted from the bottomwith the substrate. The feed rate at the beginning preferably may be onebed volume per 5-10 h and after stabilization of fermentation the feedrate can be regulated between one BV/1-5 h depending on the alcoholcontent and sweetness desired in the product. The alcohol content canvary between 0.2-2.0% w/w in the beverage To maintain the viability ofyeast, small amounts of ammonium phosphate (10-100 ppm) can be added asa nutrient.

The production can be carried out at room temperature with theparameters described above. Immobilized reactor can also be operatedunder pressure at lower temperature. Then the carbon dioxide formed isdissolved and final addition of carbon dioxide can be avoided.

In addition to the foregoing substrate other raw materials can also beused as substrates. All contain fermentable sugars, e.g., malt, fruitsand berries. The flavor of the final product will depend on rawmaterial, sugar concentration and fermentation rate.

EXAMPLE 5 Demonstration of Yeast Immobilization On Several Carriers

A brewer's yeast from the "Collection of Industrial Micro-Organisms atthe Technical Research Center of Finland" (Collection No. A-75050) wasimmobilized on two resins with anion exchange functionality.

The resins were: Granulated DEAE cellulose trademarked as SPEZYME GDC220 and a synthetic anion exchange resin having the trademark DUOLITE A568.

The immobilizations were performed according to the following procedure:

The yeast was incubated for 48 hours in a malt extract broth at 30° C.The resin was sterilized by washing with 1 M NaOH, buffered to pH5.0-5.1 and washed with sterile water. 10g dry weight of the resin wasflushed in a 20 mm i.d. glass column equipped with a glass sinter bottomplate. 100 ml of the yeast suspension was passed by gravity through thecolumn at an approximate rate of 3 bed volumes/hour, after which thecolumn was washed with 100 ml of sterile water.

The cell concentration of the yeast suspension before and after theimmobilization was determined by an agar plate count. From thedifference, the number of immobilized cells was calculated. The resultsas immobilized cells per gram of resin were as follows:

    ______________________________________                                        Resin       Yeast      Offered  Immobilized                                   ______________________________________                                        SPEZYME ®                                                                             A-75050    2,3 × 10.sup.8                                                                   2,1 × 10.sup.8                          DUOLITE ®                                                                             A-75050    2,3 × 10.sup.8                                                                   2,2 × 10.sup.8                          ______________________________________                                    

What is claimed is:
 1. A method of producing an ethanolic product in aprimary fermentation comprising:obtaining a reactor loaded with anaqueous mixture of a substantially noncompressible positively chargedcarrier having anion exchange properties; eluting the loaded reactorwith an aqueous mixture of yeast broth to cause the yeast to becomeimmobilized through electrostatic forces on the carrier; passing anaqueous substrate containing a fermentable sugar through the reactorcontaining immobilized yeast cells to produce the ethanolic product. 2.A method for producing an ethanolic product in a primary fermentationcomprising:loading a reactor with an aqueous mixture of a substantiallynoncompressible positively charged carrier having anion exchangeproperties, to produce a loaded reactor; sterilizing the loaded reactor;eluting the loaded reactor with a yeast broth to cause the yeast tobecome immobilized through electrostatic forces on the carrier; passingan aqueous substrate containing a fermentable sugar through the reactorcontaining immobilized yeast to produce the ethanolic product.
 3. Amethod according to claim 2 further comprising recovering the eluantfrom the passage of the aqueous substrate through the reactor, therebyproducing the ethanolic product.
 4. A method according to claim 1 or 2wherein a consumable ethanolic product is produced and the aqueoussubstrate is a fruit or vegetable juice, a fruit or vegetable extract, acereal grain extract or wort, hydrolyzed plant material or a sugarsyrup.
 5. A method according to claim 1 or 2 wherein the carriercomprises a continuous, porous matrix, or dimpled or reticulated, porousparticles, the matrix or particles having a structure formed from aloosely associated plurality of microparticles or microfibers which arechemically, adherently or mechanically bound together at least at somecontact points between the individual microparticles or microfibers. 6.A method according to claim 5 wherein the microparticles or microfibersforming the particles are composed of an anion exchange resin.
 7. Amethod according to claim 5 wherein the microparticles or microfibersare composed of an anion exchange resin selected from the groupconsisting of native or regenerated cellulose derivatized to provideanion exchange character, a -phenol-formaldehyde anion exchange resin,an agarose anion exchange resin and a dextrin anion exchange resin.
 8. Amethod according to claim 7 wherein the anion exchange resin is a nativeor regenerated cellulose that is derivatized to provide anion exchangecharacter.
 9. A method according to claim 7 wherein the microparticlesor microfibers are bound together by adherent links.
 10. A methodaccording to claim 7 wherein the carrier comprises particles formed frommicrofibers agglomerated with polystyrene and the anion exchange resinis diethylaminoethylene substituted cellulose.
 11. A method according toclaim 1 or 2 comprising passing the aqueous substrate through aplurality of reactors connected in series by means for removing gas fromthe fluid eluted from each reactor.
 12. A method according to claim 1 or2 wherein the aqueous substrate is passed through the reactor at apressure sufficient to maintain in a dissolved state a substantialportion of the carbon dioxide produced.
 13. A method according to claim12 wherein the pressurization is substantially maintained throughout theproduction and subsequent steps, thereby producing a carbonatedethanolic product.
 14. A method according to claim 1 or 2 whereinindustrial ethanol is produced.
 15. A method according to claim 1 or 2wherein consumable ethanol is produced.
 16. A method according to claim1 or 2 wherein the feed rate and reactor residence time are adjusted soas to provide an ethanolic product with an ethanol concentration of fromabout 0.05 to about 15 percent by volume relative to the total volume ofthe product.
 17. A method according to claim 1 or 2 wherein the flow ofaqueous substrate through the reactor is in a direction against theforce of gravity.
 18. A method according to claim 1 or 2 wherein theflow of aqueous substrate through the reactor is periodically reversed.19. A method according to claim 1 or 2 wherein the carrier is acontinuous matrix or a multitude of dimpled or reticulated, porousparticles.
 20. A method according to claim 1 or 2 wherein the reactor isa column.
 21. A method according to claim 1 or 2 further comprisingregenerating the fermentation capacity of a reactor with spent,unproductive yeast by removing the unproductive yeast from the carrierpacked within the reactor, sterilizing the packed carrier, and passingnew yeast broth through the reactor to cause new yeast cells to becomeimmobilized upon the carrier.